Science: How to run a nap-25 column

Silliness:  Why write this article?

The answer to the above question is:  for fun!  This article is at once quite silly.  Those who need to run nap-25 columns already know how to do it, or they can find out in a much more handy way by reading GE Healthcare’s awesome protocol here.  The only ones who might not know how to run a nap-25 column are those who are not scientists, and most of them won’t care to know!  Nevertheless, in case you fall into the category of non-scientist who would like to know about some aspects of esoteric science, I write this note to you!

What is a nap-25 column?  Why use one?

Nap-25 columns are used to purify DNA.  In our case, we buy thiolated DNA from Integrated DNA Technologies (IDT) – DNA that has a disulfide in it – then reduce this DNA with DTT.  A small molecule linker comes off, and this linker and the DTT must be removed from the DNA before we attach the DNA to gold nanoparticles.  Thus, we use the awesome nap-25 column.  (There is also a nap-5 column.  Nap-25 columns are used to purify larger amounts of DNA, up to but not exceeding 70 nmol of a ~33 nucleotide sequence.)

Using a nap-25 is as easy as 1-2-3!

First, to use a nap-25 column, you…

  1.  Connect said column to a ring stand with a clamp.
  2. Cut off the tip of the column at the end so the buffer can begin to drain.
  3. Take off the cap.

Then, let the buffer drain from the column and flush it three times with the appropriate buffer with which you want to elute the column – in our case – Nanopure water.  I mark the column with a permanent marker with tick marks to indicate which wash I am on so I don’t forget.  To run a sample through the column, you…

  1. Add 1 ml of DNA sample and allow it to enter the column completely.
  2. Add 1.5 ml of Nanopure water and allow it to enter the column and drip through completely.
  3. Elute the DNA with 2.5 ml Nanopure water and collect the liquid.

You can then measure the absorbance at 260 of the DNA on a Nanodrop instrument to find the concentration.  Done!

Adding the sample to the column:  two fluorescent DNAs as examples

When the DNA has run half-way through the column, you can still see it due to the DNA’s color

 

Eluting the DNA

Family: Thanksgiving at the Cuneo’s

I had such a fun time at the Cuneo’s today for Thanksgiving!  The Cuneo’s are my sister-in-law’s family.  She is the eldest of 12 and has a four year old brother!  It was so neat to be able to share a table with that many people all at once.  How special!  The turkey was smoked and extremely tasty.  One was plain and one was wrapped in bacon.  There was every kind of food imaginable there, even home-cooked pumpkin pie!  Isaiah did so well and didn’t cry at all.  He slept on the way to and from also.  I was extremely impressed and so glad he could enjoy the company!

 

Family: List of Thanks

On Thanksgiving, it seems the perfect time to list the things for which one is thankful.  I feel I have too many to count, but I shall try.

Thanksgiving:  What Am I Thankful For?

  1. Jesus:  I have real reason to be thankful for my Lord. Not only does He provide for me all the things I have and am thankful, but He allowed me, a mere nobody, to know I am loved. That is my identity – that I am loved by Him.  Without that, my life would be a dry husk that doesn’t make any sense.  I never expected to know that.  “Jesus loves me,” were just words on a page to me until he emblazoned them onto my heart in 2015.  I found He loves not just me but every person uniquely.  I am so grateful to be His handiwork, his co-laborer and His daughter.  I didn’t think I was important enough for Him to really care about or notice me.  But, I found out, He is SO great, that He notices us who are so small.  Because He lives in my life, I have hope, excitement for the future, and a hunger to know Him more.  I wonder at the great adventure He has in store for John and I and am excited to live it!
  2. John:  John daily loves me through watching Isaiah, his warm hugs, doing things for me and our lovely chats about books.  His love and support are a constant I can depend upon.  He supports me in everything I do.  When I’m worried or scared, he prays with me and lends me strength through just being there.  He gave up his career for me.  Without John, I would be lost.  I love him to the depth of my soul and I wouldn’t be the same without him.
  3. Isaiah:  Isaiah has taught me so much, even in his short 11 months of life.  He taught me how selfish I really am, and made me trust God more.  He’s also given me a window into the world of childhood and allowed me to be a kid again.  Being a mom is not glamorous.  It is hard work, and not very fun most of the time.  But I wouldn’t trade it.  I want this.  I want Isaiah in my life and I can’t wait to see who he will grow into.
  4. Mom:  Mom watches Isaiah for me every day while I am at work.  It is a labor of love.  It is a lot of hard work!  Everyone with a baby knows it.  Because she watches him, and not a daycare, I can rest in peace, knowing he is being watched by someone I trust and who loves him.  It also gives me more flexible hours with which I can work.  Mom also comes over to my house and help me clean it on a semi-regular basis.    Mom does dishes and cleans the cat litter pans.  Mom also makes dinner every night and lets me eat some.  I would be lost without my mom.  She is a pillar in my life for good, growth and strength.
  5. Dad:  Dad mows our lawn sometimes during the summer, helps my mom with Isaiah before I come over, and is a great entertainer and granddad.  He will be Isaiah’s only living granddad.  I am so thankful that Isaiah still has a granddad to love and he does love him.  Isaiah finds dad or “Opa” as we call him, infinitely entertaining and funny.
  6. Khalid:  Khalid makes this list in a big way.  I consult him about my experiments and he is always brimming with ideas.  I am constantly amazed at his knowledge base – it’s very inspiring.  I hope that I can build one as extensive.  Khalid is to me at work what my parents are to me at home.  I am so honored that I get to collaborate with someone so ingenious.  Talking about science and batting ideas back and forth with him is one of my favorite pastimes.  It’s so mentally stimulating, engaging and fun!  Khalid is also extremely supportive of me, of John, of Isaiah and my career.  I appreciate that he understands when family matters have to trump work, and doesn’t get upset.  With him at the helm, I can know that my future in science is in good hands, the lab is in good hands, and I can focus on my experiments in peace knowing all is right with the world.
  7. Brenda:  My friend and mentor Brenda Harmon helped shape me into the person I am today.  She was my organic chemistry lab teacher, but taught us how to think by making our own protocols and how to be true scientists.  She is the reason I fell in love with the chemistry behind the biology.  And she is the reason I didn’t quit science when it got down near impossible to keep going.  Without her, I wouldn’t be in grad school now.  She continues to be my best friend, one of my biggest supporters, and I love her to death.
  8. Pam:  There is no person I know like Pam Martin.  She has the most beautiful spirit in the world and I am so lucky to call her friend.  She prays with me about anything and everything in life, and I share and talk about with her all my deep ponderings on life and hear hers in turn.  We share our dreams, our fears our joys.  With her prayers behind me, I feel protected and loved.  She is such a support!
  9. Graduate school:  I cannot tell you what graduate school means to me.  I am so thankful to be in it, getting the opportunity to pursue my dream of being on the cutting edge of knowledge and even getting paid for it!  That is all I have ever wanted:  not a specific position (like being a PI), not a specific place (like Berkeley or MIT), not even the degree itself.  I just want to be on the cutting edge of knowledge as long as I can, and I am doing that.  My most intense moment of euphoria in my whole life was the Emory visitation weekend, when I was listening to Khalid describe the project I am working on now and have helped make a reality.  It was in that moment that I realized that what I had been working toward so long – doing research – I would finally be able to do!  I could finally study and experiment whatever I wanted!  I had freedom!  I could do real research!  I was a valuable, recruitable resource!  My dreams were becoming reality!  I already have my dreams, even if I never get anything else.  Graduate school has also given me friends.  I didn’t have  a lot of friends growing  up, being shy, and none in college, because I studied too much – but now – in grad school, I actually have friends in my classmates, and it has been a real joy to share life with them.
  10. ARCS:  The ARCS scholarship is an incredible blessing.  It has allowed me to stay afloat, with a baby in tow.  It was extremely validating.  I felt such relief, that I could be among such an elite crew of people honored with the award.  My sponsors Sara Jean and Fred Burke are wonderful and amazing people.  I am so blessed to know them.  Their support has meant the world to John and I.  It has also been awesome to get to go to events where I can network with some truly awesome scientists, feel honored and valued, for who I am and what I do.  Those moments can be rare in science.  And I value every one.
  11. My first paper:  My first paper was recently published as a “just accepted” manuscript in ACS Chemical Biology, as I am sure everyone knows, as I cannot stop talking about it.  Having a first author paper has been a dream of mine ever since I was a freshman in college.  Now, thanks to everyone’s help and support, it has become a reality.  I cannot describe my joy at seeing my work for the last four years in print, not to mention that, at least 60% of the paper is completely my original words!  Khalid helped shape the words, and he changed some things, but much of the writing stayed true.  He did not completely re-write it.  It remains my own.  That makes me so proud.
  12. My house:  I love our house.  John and I waited a long time before buying one, and we bought one large enough to support multiple kids in the future.  I am so grateful to have a place to call my own and to raise a family in.  I never dreamed I would own a place like our home that is so fancy-looking and great!  I love it.  It’s perfect for us.
  13. My computer(s):  I have a desktop and a laptop.  Both these computers were purchased to be top-of-the-line and fast.  I love fast computers.  Most everything I love doing is on a computer:  writing, blogging, reading science articles, building websites, some minor gaming when I can – it’s my connection to knowledge and to social media and the rest of the world.  Therefore, I love my computers and am so thankful for them every day.
  14. My cats:  I have always lived with cats but never had my own until recently, when we purchased three orange kittens in 2016.  Having an orange kitten in particular was a childhood dream of mine.  Our cats also taught me responsibility, similar to having a baby, but less work.  Still work though – having my own cats is a lot different than growing up with them.  They are more special and also more work!  And I do love them dearly.  I can tell them apart by their meow’s and they each have their own unique personalities.
  15. Books:  I love books.  I’m glad they exist.  I love reading them, especially about God, and getting transported into other worlds not my own.  I love trying to write books.  I love collecting them and putting them on shelves.  Books are all around just awesome.

This list is just a sprinkling of the things that are dearest to my heart, that I think about all the time and for which I am most grateful.  I wouldn’t be who I am today without these people and things.  Thank you from the bottom of my heart for being a part of my life – every one of you – whether I know you in passing or not.  I am so grateful for each and every one of you.  Life is so much more exciting when it is shared.

Science: My first paper goes live!

Site-Selective RNA Splicing Nanozyme
DNAzyme and RtcB 
Conjugates on a Gold Nanoparticle

Table of Contents Graphic (TOC) for my paper: Shows an intron (small segment) being removed from RNA by a nanozyme composed of a gold nanoparticle with attached DNAzymes and RtcB.

Hello world!  

(In this post, I  use blue notes to describe details of my science to an educated non-science audience.)

I am overjoyed to announce the going live of my very first scientific article in the journal ACS Chemical Biology!  It can be found here in PubMed or here at the ACS Chemical Biology website (1).

I have waited SO LONG to tell you all about my work!  I have been working diligently on this system (with some side projects as well) for about four years.  

In it, I describe the construction and activity of my RNA splicing nanozyme!  It is built using 10-23 DNAzymes and the E.coli ligase RtcB attached to a gold nanoparticle scaffold.  Note that this nanozyme only removes introns.  It cannot add exogenous RNA into a strand.  The complete nanozyme construct functions currently at 10% efficiency.  However, we have some plans to boost that yield in the future.  When used in excess and not attached to a particle, the enzymes themselves can achieve up to 66% yield or higher in some cases (data not shown).  RtcB is the limiting reagent, as it is likely to be a single-turnover enzyme in E.coli.  The amount of RtcB on the nanozyme is also far less than the attached DNAzymes:  only between 1-5 RtcB per particle and between ~50-100 DNAzymes per particle.

What is a 10-23 DNAzyme?

DNAzymes are short pieces of DNA about 33 nucleotides long that can act like enzymes.  The particular DNAzyme I am using in my system works enzymatically by cutting RNA strands.  It can be targeted to cut anywhere along a particular RNA target.  An RNA ligase is a protein enzyme that joins RNA strands together.  By coupling these two enzymes with each other on a gold nanoparticle, RNA “scissors” and RNA “glue,” this nanoparticle machine, nanomachine or “nanozyme” can effectively cut up RNA and paste it back together.  In my paper, I use its activity to remove a short segment from an RNA strand.

Two 10-23 DNAzymes (DZ) are shown at left.  These are the ones I used.  The blue section is the catalytic core, responsible for the enzymatic activity of the DNAzyme.  The core chelates (or latches onto) a manganese ion, which assists in shaping and activating the core to perform its catalytic function of cutting RNA.

 

What is RtcB?

Shown is a ribbon structure for P. horikoshii RtcB [PDB=4ISZ].
 RtcB is a protein – more specifically – an enzyme called a ligase that ligates (or joins) RNA molecules together.  It is the “molecular glue” of my nanozyme.  It is thought to be involved in RNA repair in E.coli.  In mammalian cells, it acts differently than the E.coli protein (and also has another name – HSPC117).  It associates with other proteins such as archease (2,3) and DDX1, is multi-turnover (2) and is involved in tRNA splicing in mammalian cells (3).  It is also involved in upregulating the unfolded protein response (4).  We chose RtcB as the ligase for our nanozyme because it can specifically act on DNAzyme cleaved ends (2’3′-cyclic phosphates and 3′-OHs), ligating them directly, rather than the longer “healing and sealing” mechanism preferred by other ligases.

What is the end game?  Why does this nanozyme matter?

The home run goal for this nanozyme would be for it to be used to correct genetic defects in RNA inside cells – though that has still to be realized.  It is also the first example of DNAzymes collaborating with a ligase for RNA splicing, and a completely new method of manipulating RNA in vitro and potentially one day, inside cells.

With much thanks!!

I cannot mention this project without thanking the people involved who helped make it happen.  At the top of the list is my PI, adviser and boss Khalid Salaita.  He is the most amazing mentor I could hope for and has been a cheerleader behind me all the way, through the long process of trouble shooting and getting this nanozyme working.  Oh the stories I have to tell!  Also high on the list is Kevin Yehl, who originally thought of the idea of combining DNAzymes and RtcB, but didn’t have the time to work on it.  So, I took up the torch and carried it forward.  I often turned to Kevin, during the years that followed, for advice and help interpreting data.  He’s known as a wizard for good reason.  Then, there’s Kornelia, who helped make a batch of cysteine labeled RtcB for me, Brendan, who helped me with flow cytometry showing cell uptake of the nanozyme, and Roxy, who did all the FLIM experiments needed to show that RtcB is bound to the gold surface.  Then, there are the unsung heroes like Yun, who I love to death.  She was my special “stress friend” my first three years as a graduate student, meaning that when I was stressed, I would find her and cry on her shoulder.  She also taught me loads about how to carry myself and present better.  She now lives in China with her husband.  Yuan was also another special friend who supported me undyingly during my work and I so appreciate her kind words, encouragement and random cookies she would give me.  Thank you to all you amazing guys!!  Without you, I wouldn’t have been able to endure to see this project through.

Questions?

If you have comments or questions please email me!  I absolutely love talking about this work.

References

1. Petree, Yehl, Galior, Glazier, Deal, Salaita. DOI: Site-Selective RNA Splicing Nanozyme: DNAzyme and RtcB Conjugates on a Gold Nanoparticle. ACS Chemical Biology. DOI: 10.1021/acschembio.7b00437

2. Desai, Beltrame, Raines. (2015). Coevolution of RtcB and Archease created a multiple-turnover RNA ligase. RNA. http://www.rnajournal.org/cgi/doi/10.1261/rna.052639.115.

3. Popow, J., Jurkin, J., Schleiffer, A., & Martinez, J. (2014). Analysis of orthologous groups reveals archease and DDX1 as tRNA splicing factors. Nature, 511(7507), 104–7. http://doi.org/10.1038/nature13284

4. Kosmaczewski, S. G., Edwards, T. J., Han, S. M., Eckwahl, M. J., Meyer, B. I., Peach, S., … Hammarlund, M. (2014). The RtcB RNA ligase is an essential component of the metazoan unfolded protein response. The EMBO Journal, 15(12), 1278–1285.

Faith: A song that touched me

Walking around these walls
I thought by now they’d fall
But You have never failed me yet
Waiting for change to come
Knowing the battle’s won
For You have never failed me yet

Your promise still stands
Great is Your faithfulness, faithfulness
I’m still in Your hands
This is my confidence, You’ve never failed me yet

I know the night won’t last
Your Word will come to pass
My heart will sing Your praise again
Jesus, You’re still enough
Keep me within Your love
My heart will sing Your praise again

Your promise still stands
Great is Your faithfulness, faithfulness
I’m still in Your hands
This is my confidence, You never failed

Your promise still stands
Great is Your faithfulness, faithfulness
I’m still in Your hands
This is my confidence, You never failed me yet

I’ve seen You move, come move the mountains
And I believe, I’ll see You do it again
You made a way, where there was no way
And I believe, I’ll see You do it again 

I’ve seen You move, come move the mountains
And I believe, I’ll see You do it again
You made a way, where there was no way
And I believe, I’ll see You do it again 

I’ve seen You move, come move the mountains
And I believe, I’ll see You do it again
You made a way, where there was no way
And I believe, I’ll see You do it again 

I’ll see You do it again
Oh-oh

Your promise still stands
Great is Your faithfulness, faithfulness
I’m still in Your hands
This is my confidence, You never failed

Your promise still stands
Great is Your faithfulness, faithfulness
I’m still in Your hands
This is my confidence, You never failed me yet

And You never failed me yet
I never will forget
You never failed me yet
And I never will forget

Status: A Crazy Day

The experiments!

Today, Brendan showed me how to add DNA to my mammalian cells with lipofectamine.  I was also trying to spin down more particles at the same time, and make my own media for the cells.  Finally, I got the transfection going.  Then, I worked on spinning down and washing my particles for a kinetics assay.  These nanoparticles have a DNAzyme attached to them and I wanted to test their catalytic activity.  I did this by having the DNAzyme cleave a FAM / BHQ RNA / DNA hybrid substrate.  Essentially, what this means is that the DNAzyme would cut the substrate, releasing the two halves into solution.  When the two halves were released, the fluorophore (FAM) would glow more brightly because it was no longer near the quencher (BHQ).  Therefore, the activity of the DNAzymes can be measured as fluorescence over time.  I also added a toxin to some of my cells.  Why? Because.  It was necessary for my experiment.  

Fast paced all round!

Today was really fast paced.  I was constantly moving.  I got just enough time to eat lunch (leftover pizza) during one of the spins I was doing on my nanoparticles.  I noticed Khalid seemed extra busy today.  But he’s seemed busy all week.  However, today, loads of people were trying to talk to him constantly.  I made sure to leave him alone.  He has 17 students to watch over plus rotation students.  That’s just a lot of work. 

Tomorrow!

Tomorrow, I am doing flow cytometry sample prep from 8 a.m. – 10 a.m.  Then, I am doing flow cytometry training from 10 a.m. – 1 p.m.  Then, we have the nanozyme subgroup meeting at 1:30 p.m.  Then I have to do a spin of 70 tubes of nanoparticles for our collaborator.  Finally, I’m going to eat dumplings with Alisina, to celebrate his passing his second year report because dumplings are my favorite.  We are going to go to a dumpling restaurant!  Total fun.  More soon!

Keywords / Terms:  nanoparticles (NPs, 13 nm gold nanoparticles; that’s the size of viruses), DNAzyme (deoxyribonucleotide, a ~33 nucleotide piece of DNA that can act like an enzyme by cutting RNA), fluorophore (a dye molecule that glows), spins (centrifuging something), lipofectamine (a transfection reagent), transfection (adding DNA to mammalian cells), flow cytometry (instrument that measures the fluorescence of cells and can count them for you)

Family: Stories of Isaiah

There’s a lot that goes into caring for a baby.  I had no idea the magnitude of this when I was pregnant.  It involves near continuous diaper changes and feedings, and then entertaining the baby throughout the day.  I related some of that story on my previous website in the Family blog here.  Ever want to know what it’s like to live with a 10 month old?  I did before I had him.  Here is a day in the life of Isaiah. 

Weekday

Through the week, 7 am – 7 pm my mom cares for him – most of the day.  I am extremely fortunate and blessed that she does this for me.  It allows me to go to work with peace of mind.  We used to have a camera on him during the day, that I could pop onto and watch him while I was at work.  It has since been sitting in his room though and I keep forgetting to bring it back to my mom’s so she can start it up again.

Bedtime Routine

When I get home, it’s usually 7 or 7:30  pm, and I come home to mom and dad’s where Isaiah is, hold him and play with him some, and eat dinner with my parents.  They kindly provide the opportunity for me to eat dinner with them 99% of the time.  I feed Isaiah his dinner – usually either a bottle, some baby food or both – then bring him home around 8 p.m., at which point he goes to bed.

When I take Isaiah to bed, I have a routine I do with him every night.  I swing him into bed, counting to 10 in German.  This is something my mom used to do with me when I was little.  I can still count to 100 in German.  I then read him 10 verses of a Psalm from the KJV Bible.  He ignores this — rolling around in his crib and exploring — but I like to do it.  Perhaps I’ll switch to a more kid friendly translation someday when he’s older.  I was hoping he could get used to the KJV if I read it to him.  It was what I grew up on and I became fond of it.  I’m not a die-hard though.  I read other versions.  We are up to Psalm 88 currently.

Then, I lay him down in bed (since he’s been crawling around) give him his blanket, and always say the following to him (more or less):

“Isaiah it’s time for sleeping now!  Remember, Jesus loves you very much.  He made you.  And I love you.  And your Dad loves you.  And your Oma and Opa love you.  And your Grandma Jackie loves you.  And your Aunt Elizabeth and Uncle James love you.  And your Aunt Casey and Uncle Matt love you.  And we’re all going to take good care of you!  Now, sleep well!  I love you very much Isaiah.”

He always smiles at all the “I love you’s” and seems to listen.  Most days, he goes right to sleep or at least doesn’t make a peep when I turn out the light and close the door.  I’m so lucky.  We had a spate of time where he would cry when we left, and we had to sit by him until he settled down for 5-10 min before it was safe to leave.  However, most of the time, he’s doing pretty good again.  I count that a huge blessing.  Soon, I hope to be reading him a bedtime story every night too.

Saturday:  A day with Isaiah and what we do together

Saturday is the day I watch Isaiah full time.  He usually lets me sleep in until 8 a.m.  Then, I’ll roll out of bed and feed him.  Isaiah loves to crawl and explore everything.  He also loves to go places he shouldn’t.  Favorite activities include stuffing his mouth full of carpet fuzz that he finds pieces of on the floor, or going for the cat scratching posts and eating the cardboard pieces off the floor, or playing with the cords hanging out of the lamp or the Playstation.  He once got interested in the cat pheromone plug-ins I have in the wall.  I tried to get him away from those and at last succeeded.  He also has safe toys.  He likes his cars the best, rolling them around on the floor.  Isaiah is also constantly babbling.  He loves practicing talking.  It’s kind of cute.  He says “cat” pretty well, and I wonder if he connects the word “cat” with our cats yet.  He likes flipping through books too – especially if he can turn the pages.  He loves turning pages.

If he is in the kitchen, he’ll typically make a beeline for the cat food bowls and the litter pan.  If he is in the dining room, he loves the computer cords, and also sifting through the mail and eating it.  John has a box of sprue – plastic Warhammer model pieces in a box – and Isaiah loves to go over to it, take them all out and throw them everywhere.  

Sometimes, he gets frustrated at a toy or if a wire doesn’t move like he wants, and he’ll cry.  He’s a pretty good eater.  We started him on vegetables first.  I admit, though I like the idea, I haven’t really gone with baby led weaning, because it’s not the way my mom and dad grew up and while – I wouldn’t mind experimenting – my mom, who watches him 90% of the time, can’t stomach the idea.  I don’t feel like I can do it without her support.  However, Isaiah has had plenty of fruit in a mesh feeder.  He won’t eat any beef baby food though – particularly the Gerber kind I used to like as a kid – nope – he hates it.  Won’t open his mouth at all once he’s tasted it.  But he eats peas fine, he’ll stomach green beans and his favorite is carrots, ironically enough.  He also likes fruits.  He’ll eat mixed fruits and mixed veggies.  There’s nothing really he refuses except beef.

Sometimes, we’ll go on walks in the stroller – though we’ve done that less of late.  We used to do it all the time when he was younger.  While watching Isaiah, I’ll often turn on a movie.  Or, if I’m brave, I’ll try to get house work done:  the dishes, cleaning the floor from puffs he’s dropped (he loves puffs! – it’s a cereal for babies), the laundry.  I try to carry him with me to the laundry room and bed room when I do the laundry.  

John gets home about 4 p.m. and often John will help watch him at that point – he’s so sweet like that.  Isaiah loves playing with John.  John is great at making baby talk and baby noises with him that make Isaiah laugh. 

On Saturdays, I usually always give Isaiah a bath.  I still give him baths in the sink using a baby bath tub.  I tried the bathroom tub, but it breaks my back.  Painful.  A lot of times on Saturdays, mom and dad will come over and make breakfast and help me clean the house, which is a HUGE blessing, let me tell you!  I can’t describe how appreciative I am of all they do for me constantly.

Sunday

On Sunday, John lets me sleep in and he feeds Isaiah instead of me.  I’ll roll out of bed about the same time though around 8 or 8:30 a.m.  We usually make Pillsbury crescents or rolls for breakfast.  Then, we go to church.  John will come – or if he has sinus trouble or a bad week at work – he’ll stay home – and I’ll take Isaiah to church with me.  I give him to Brittany, the usual nursery watcher, and she knows all the things to make him calm. When he was younger, he didn’t make a peep in the nursery.  He would always sleep and was such a good baby.  Lately though, he’s been a more fussy customer.  However, he likes music.  There’s a favorite musical toy they give to him that makes him stop crying.  He also like it when Brittany will take him out into the halls and walk up and down with him.  He likes to be held and / or moving at all times.  If she puts him down, he won’t last for more than a minute without getting upset.  He also likes to talk to her badge, which she thought was funny.

After church, we will usually eat lunch with Jackie Petree.  Then, most times, Jackie will watch Isaiah from 12 – 4:30 pm and this fact allows me to finish the house work – scoop the cat litter pans – which I do once a week, and finish the laundry, fold it and iron John’s shirts for the week.

And that is a usual week with Isaiah!  More soon!