ABCDE
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Opioid Project Mutagenesis
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DETAILS TO BE ADDED AS PROJECT PROGRESSES
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Questions? Email Courtney Karl at cek5120@truman.edu. Also, see detailed schedule on google drive.
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InitialsDate CompletedTaskNotes
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JM2/8KCNJ3 sequencing primers designedon sequencing primers master list
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KB2/9KCNJ9 sequencing primers designedon sequencing primers master list
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?2/16KCNJ3, KCNJ9,and OPRM1 plasmids ordered
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SB2/27Bacterial plates of OPRM1 and KCNJ9 placed in incubator at 2:30.
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YN2/28Bacterial plates of OPRM1 and KCNJ9 placed in Fridge B at 11:00 am
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GP3/19Streak was made on agar plate labeled “2/23/18 FS LB + Amp” using KCNJ9. KCNJ9 was procured from the -80 freezer and returned after use. Bottle label for KCNJ9 is 27250. Placed plate in incubator. -GPWas streaked on wrong plate
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SB3/20KCNJ9 was restreaked on the correct plate and placed in the incubator around 3:45.
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JA3/21Dried DNA pellets from MiniPrep resuspended in tubes labeled ‘OPRM1-2 and OPRM1-1. Resuspended in 20 ul of MBG water.Labelled with orange stickers and placed in Freezer C, Box 17
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EM3/27Mini prep was performed on two samples of KCNJ9
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FS3/28hMOR insertion and deletion primers diluted to PCR specifications and placed in box 18 in freezer C. PCR began. Placed in box 18
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EM4/24Ran gel with 1uL in each lane of KCNJ9 1, KCNJ9 2, OPRM1 1, and OPRM1 2
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JMM4/26KCNJ3 and KCNJ9 sequencedKCNJ9 missing stop codon; KCNJ3 is complete
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JMM5/1 Made construct sequences for KCNJ3, KCNJ9, and OPRM1 in their respective vectors. Primers for subcloning can now be made
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2019
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CK1/14Found concentrations of KCNJ3, KCNJ9-1&2, and OPRM1-1&2 with NanoDropNanodrop concentrations saved under DNA and RNA inventory
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MP1/15Ran gel mp011519 with diluted OPRM1-1 and -2, KCNJ3, KCNJ9 1 and 2mp011519 pic link
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CK1/15Found sequencing primers for OPRM1 and the forward sequence of KCNJ3. Diluted OPRM1-1 and OPRM1-2 into aliquots with a target concentration of 1000 ng/ul. Ran gel of all opioid plasmids.Need to design KCNJ9 primers and KCNJ3 reverse primer. Need to NanoDrop OPRM1-1 and OPRM1-2 aliquots. Need to view/analyze gel. Sequencing Primer Data/Visuals saved on G drive in opioid project folder. visualize primers with APE software
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CK1/17ordered opioids Hydrododone and DAMGO. Made Snap gene plasmids of KCNJ9 and OPRM1Need to finish KCNJ3 Snap gene Plasmid. Checked against uniprot.
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CK1/21Designed sequencing primers KCNJ3 R1, KCNJ9 R1, KCNJ9 F2see sequencing info under opioid project folder on google drive
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CK1/22Ordered sequencing primers KCNJ3 R1, KCNJ9 R1, KCNJ9 F2order form saved under opioid project folder, added sequences to Sequencing Primers Master List, still need to do stuff listed aboove in notes
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CK1/24Prepared plasmids and primers for sequencing Nanodrop OPRM1 1 & 2 aliquots. Hopefully, send samples off for sequencing. Finish KCNJ3 Snap gene plasmid, check OPRM1 snap gene plasmids. Look at gel concentration results
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AS1/24Prepared and measured concentrations of KCNJ3, KCNJ9-1, KCNJ9-2, OPRM1-1, OPRM1-2, and primers OPRM1 F2, KCNJ3 F3, and OPRM1 R1 with Nanodrop.
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CK1/29Samples calculations ready for thursday
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CK1/31Sent samples for sequencing! YAY! Also made sequencing instructions for future referenceSaved order form under opiod project folder on G drive. Sequencing instruction in ooioid folder on google drive. Still need to finish KCNJ3 Snap gene plasmid, check OPRM1 snap gene plasmids, and look at gel concentration results
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CK2/4Ananlyzed sequencing results for KCNJ9-1 and 2 and KCNJ3.KCNJ3 missing stop codon, KCNJ9-1 end did not sequence connection between plasmid and protein coding sequence, KCNJ9-1 beginning has and extra T after bp 940 (near end of sequence though), KCNJ9-2 beginning does not confirm either, KCNJ9-2 end gave no relevant info. Still need to look at OPRM1
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CK2/15Analyzed sequencing results for OPRM1-1 and 2OPRM1-1 and OPRM1-2 beginning sequencing results showed that the linker sequence is actually GTACAAAAAAGTTGGCACC versus GTACAAAAAAGCAGGCTCCACC. OPRM1-1 and OPRM1-2 end both have stop codons missing- TTG intead of TAG
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CK2/19Analyzed OPRM1 and KCNJ9 sequences
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CK2/26Designed mutagenesis primers to fix OPRM1 stop codon, made OPRM1 snapgene with bad stop codon, made word doc with all alignmentsNeed too do deletion of chunk inbetween T7 promoter and protein coding sequence start on OPRM1
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CK3/19Figured out what primers need to be ordered in the process of making plan. KCNJ9 sequence from 4/18 had correct stop codon so no longer need to resequence. Yay!Check out plan in opiod project folder on G drive for more info
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CK3/26Designed Q5 mutagenesis primers for KCNJ3 and OPRM1 stop codons, IRES removal from OPRM1 and KCNJ3, and KCNJ9 T7 insertion primers. Filled out pink order request form for NEBuilder HIFI DNA Assembly master mix- 10 rxns- will be used alternate method for T7 promoter insertion.Saved in opiod project mutagenesis primers on the G drive. KCNJ3 and OPRM1 IRES removal primers are the same
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CK4/1Started working on plan with plans A,B, and C. Need to look into UTR's next time to seee if adding restriction enzymes disrupts them, especially in non pGHE plasmids
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CK4/2Designed NEBuilder HiFi DNA Assembly Kit primers to insert a T7 promoter into KCNJ9. Made XmaI restriction site substitution primers for KCNJ9. Looked up Kozak's sequence in KCNJ9 plasmid and found GCCCACCATGG and made sure XmaI restriction site was upsteream. Still need to check Kozacks sequences for KCNJ3 and OPRM1 and design XmaI restriction sites. Also DO NOT forget to make note somewhere later on to orger oligo of T7 promoter for NEBuilderVideo of NEBuilder Primer design needs to be edited but one exists. After finishing XmaI primers look into if we need new sequencing primers. The KCNJ9 sequencing primer is too far away but in 2018 they use the backwards T7 promoter downstream of the protein coding sequence to sequence the end of KCNJ9 so need to ask Dr. Norimatsu if we can just use that instead of ordering a new one. A new one may be better though because could use to do sequencing in pGHE. Also need to look into UTR's in the future.
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CK4/16Finished designing XmaI restiction site mutagenesis primers for KCNJ3 and OPRM1. Started mutagensis primer visualization word doc for mutagenesis plan presentation. KCNJ3 Kozack sequence +4 T vs G. Need to ok with Dr. Norimatsu. OPRM1 SacII restiction site primers don't line up so need to probably redo.
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CK4/26Continued working on mutagenesis primer visualization. KCNJ9 has restriction sites for XmaI and SacII in protein coding sequence so we need to add either BtgI or NotI restriction site (need to check plasmid for still for both) after stop codon and KflI or PpuMI before Kozak's sequence (need to check plasmid still). Made OPRM1 plasmid after edits for original plasmid and pGHE. Need to design KCNJ9 end restriction site primers and redo beginning restriction site primers. Need to check if KCNJ3 beginning restriction site primers still work with IRES deletion and make the 2 edited plasmids like I did for OPRM1.
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CK4/29Continued working on mutagenesis primer visualization. Made KCNJ3 APE plasmids for after edits for original plasmid and insertion into pGHE. Q5 seems to be easier for KCNJ9 T7 insertion. Also note issues with NE builder primers. For KCNJ9 Restriction sites make one for KflI (GGGACCC) and BsteII (GGTTACC).
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CK
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LH8/14Beginnings and ends of hMOR, KCNJ3, and KCNJ9 were sequenced and confirmed; few complications with KCNJ9. Real sequences of KCNJ3 and J9 were made as well. All files kept in folder "Lucas Stuff" on desktop of both East computersThe sequence for the end of J9 from genewiz was shorter than expected. We will have to resend a sample for sequenceing after the DNA is cleaned. This will delay making the primers for these plasmids until we can sucesfully sequence all three so we will be able to order all at once
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LH8/19Primers sequenced for insertion and deletions of hMOR and KNCJ3 (LINK IN NOTES). Files for hMOR, KCNJ3, and KCNJ9 moved to respective folders in Gdrive under "Opiod Project". KCNJ9 DNA will be cleaned today and made ready for sending to geneWIZLink to Google doc. file for primers: HERE
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CK8/21Resuspended KCNJ9 plasmids and places in freezer A in red rack. Finished plan to "fix" plasmids. Need to order primers for KCNJ3 and Hifi DNA Assembly (primers on word document called New Opioid Plasmid Plan in the Opiod Project folder on the G drive).
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TR8/26Ordered stop codon and HIFI mutation primers
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need to nanodrop KCNJ9 that was purified.
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LH8/27hMOR insertion and deletion primers diluted to PCR specifications and placed in box 18 in freezer C. PCR began Labeled hMOR IN/DEL F/R. IN= insertion DEL=deletion F=forward R=reverse
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LH8/28PCR rsults sucessful for hMOR inseriton and deletion primers. link to gel image attached Gdrive-> physiology lab -> Norimatsu lab -> Lab projects -> opiod -> mu or -> OPRM1 -> LH-Gel hMOR 8-28-29
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CK8/29Resuspended KCNJ3 stop codon mutagenesis primers to a concentration of 100 uM and made 10 uM aliquots and put in box 18. Made an aliquot of the KCNJ3 plasmid at 25 ng/uL and put in box 18. Performed PCR mutagenesis of KCNJ3 stop codon and used 25 ng/uL of KCNJ3 Plasmid in the reaction mix.Forgot to keep reagents on ice when mixing. Still need to resuspend other primers.
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CK9/12Tranformed OPRM1 stop codon insertion and deletion plasmids that Lucas made. Did DnpI digestion for KCNJ3 PCR product.Don't think OPRM1 transformations worked because SOC not cloudy looking when plated. May be issue in phospyorylation or circularization of those plasmids.
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CK9/26Phosphorylated and ligated KCNJ3 PCR product. It should now be circularized for the next step which is Hifi DNA assembly.OPRM1 transformations worked whoo! KCNJ3 in Box 18 now and original PCR souce in PCR box in freezer A labeled CK still.
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CK10/17Ran gel to analyze concentrations of KCNJ9 25 ng/uL aliquot, KCNJ3 fixed stop codon 25 ng/ul aliquot, and ORPM1 fixed stop codon insertion aliquot. KCNJ9 has a concentration of 11.809 ng/uL which is good to use for PCR. KCNJ3 and OPRM1 both did not have bands show up which means they were too diluted or the PCR failed. Next, time run gel with higher concentration of KCNJ3 and OPRM1. Also reorganixed box 18 and taped key inside the lid. Also fpund the pruified KCNJ9 samples that LH purified in Freezer A and put in box 18Started to make PCR reaction tubes for all three plasmids- each has water and primers in it and they still need DNA and mastermizx added. I'm worried that leaving the primers at such a low concentration may lead to degredation so if more if they are sitting more than a week I would redo them.
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CK10/31Updated opioid project page on lab wiki and made new snapgene and Ape files for opioid project plasmids. None of our Hifi primers will work. We will use the pGHE backbone from pGHE_hTAAR1 now (see sequence on lab wiki).Will do PCR subcloning now to get protein coding sequences into pGHE.
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TR11/4KCNJ3 DNPI digest after stop codon fixDpnI FD of "KCNJ3 PCR" started at 1:30 will be done at 2:30
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TR11/4KCNJ3 Phosphorlaytion and ligationDone tube placed in box 14
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MB+AH11/5KCNJ3 and KCNJ9 transformationTransformation of KCNJ9 was "LH purified KCNJ9-2"
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MH11/6Inoculation of KCNJ3 and KCNJ9
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TR+MB11/7Maxiprep KCNJ3 and KCNJ9 stopped at ethanol step will finish tomorrow
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CK11/7Designed and ordered primers for KNCJ3, KCNJ9, OPRM1, and the pGHE backbone for PCR subcloning. The protein coding sequence primers do not incude the stop codon. The pGHE backbone primers were designed to amplify the hTAAR1_pGHE backbone, including the existing stop codon. Also updated lab wiki up to date
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MH11/8finished Maxiprep. DNA left to dry on counter
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TR11/11Resuspend KCNJ3 and KCNJ9 in 1mL of MBG waterplaced in blue rack in freezer A
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AH11/11Gel of KCNJ3 and KCNJ9saved as ah111119
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TR11/13Ordered sequencing primers for KCNJ3 middle and KCNJ9 middle and KCNJ9 end. Did not order begining primer for KCNJ9- primer given by genescript was the same as the current one. Current KCNJ9 Sequencing primers are labeled as bad?
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TR11/13Resuspend PCR subcloning primersplaced in subclonning box 15
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CK11/14Deleted old/inaccurate files in the Opioid Project file on the G drive and went through box 18. Tested out the new PureLink PCR Purification Kit on an OPRM1 aliquot and nanodropped sample using the elution buffer as a blank. Sample originally thought to be around 500 ng/uL was measured to be 61 ng/uL after purification. A260/A280 and A260/A230 values were satisfactory. Should check concentration of aliquot with gel too. Performed PCR reactions to amplify pGHE backbone and OPRM1 protein coding sequence for future blunt ended PCR subcloning- tubes need to be taken out of thermocycler and put in PCR rack tomorrow morning. Used 6 uL (and adjusted MGB water added to 4 uL) of 3.775 ng/uL TAAR1 pGHE dilute DNA in box 12 with green label for pGHE amplification. Subcloning primers are in box 15. Annealing temperatuse used for PCR was 62.3 and elongation time was set to 400 seconds.
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TR11/15Resuspend KCNJ3 and KCNJ9 sequencing primers.Placed in box 18
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TR11/18Ran gel with pGHE backbone and OPRM1, sent KCNJ3 and KCNJ9 for sequencing of the protein coding sequence
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TR11/20DpnI digested pGHE backbone and OPRM1, OPRM1 and pGHE ligated
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CK11/21Analyzed KCNJ9 and KCNJ3 sequencing results, results (see 11/15/19 sequencing results word doc in G drive -> lab projects -> opioid -> sequencing results -> 11-15-19). Requested re-run of KCNJ9_begining-KCNJ9_R1. End of KCNJ9 failed due to primer positions. A portion of KCNJ3 was not sequenced but according to trace files I think it is okay.
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TR11/22Started PCR subcloning for KCNJ9 PCR will be done at 1
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TR11/22PCR subcloning for KCNJ3 PCR will be done at 5
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TR12/2Run gel for success of PCR for KCNJ9 and KCNJ3saved as tr120219
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TR12/2DpnI digest KCNJ9 and KCNJ3 insert digest started at 3pm
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TR12/3phosphorylated KCNJ3 and KCNJ9 inserts and ligate with pGHE backbonedone at 12:10pm
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CK12/3Transformed OPRM1_pGHE, KCNJ9_pGHE, and KCNJ3_pGHE. Figured out sequencing primers for all plasmids and filled out order form draft on Genewiz. Screenshot of which primers to use is saved as pGHE Sequencing Primers Plan in Opioid Project Folder on the G drive.Slightly concerned about competent cells used for KCNJ3. They were pipetted 4 times because the aliquot did not contain the expected 40 uL.
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CF+ MB+AC+AH12/5Mini or maxi prep pGHE plasmidsminiprep left at ethanol step. Finished on 12/09/19
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TR12/10Gel of KCNJ3 , KCNJ9 and OPRM1 from miniprep
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TR12/20Send samples for sequencing
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TR1/6KCNJ3, KCNJ9 sequencing results- No priming, DNA Conc too low or Subcloning unsucessful. OPRM1 still in pANT7 plasmid, suggesting the parental DNA was not completely digested. Redo digest.
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TR1/8PCR of KCNJ9 insert. Orignal insert disposed of bands not clear. DnpI digest of KCNJ3, OPRM1, and pGHE backbone.
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TR1/9phosphorylate backbone and see if transformation is successful. Success
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TR1/13Redo transformation for OPRM1, KCNJ3, Subcloning.
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CK1/14Ananlyzed the re-run of KCNJ9_beginning-KCNJ9_R1, and the re-run turned out worse :(. Updated the lab wiki. Updated the Box 18 key. Designed opioid project Hifi DNA assembly primers (saved in opioid project folder in G drive). Inoculated 3 OPRM1 colonies and 3 KCNJ3 colonies for miniprep.
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TR1/15mini prep of Blunt end subcloning OPRM1, KCNJ3 (1-3)Drying 1/21/20. Resuspended.
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TR1/17Another mini prep of Blunt end Subcloing OPRM1, KCNJ3 (4-6)needs to be completed 1/21/20 left at RNAse step.
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TR+LC1/21HIFI subcloning primers ordered. Resuspended. Need 10uM dilution
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CK1/21Set up PCR reactions for OPRM1, KCNJ9, KCNJ3, EPG, OPRM1 pGHE, KCNJ9 pGHE, and KCNJ3 pGHE. Ran thermocycler for OPRM1, KCNJ9, and KCNJ3. OPRM1, KCNJ9, and KCNJ3 moved to freezer A PCR box. Gel next to orignal DNA for success
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TR1/22Ran thermocycler for OPRM1 pGHE, KCNJ9 pGHE, and KCNJ3 pGHE.
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TR1/23Ran gel for HIFI protein coding sequences OPRM1, KCNJ3, KCNJ9 and OPRM1 (1-3) blunt ended subcloning. KCNJ9 HIFI failed. Redo PCR and check KCNJ9 with nanodrop before preparing reaction. Lane 8 for blunt ended subcloning OPRM1 looks promising. Gel saved under tr012220
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CF1/23Completed mini prep for Blunt end Subcloing OPRM1, KCNJ3 (4-6)Left at ethanol step. In freezer A. Finished 1/27
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LC1/23Ran gel for HIfFi pGHE reactions. All successful. Gel saved under lc012220.
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TR1/24ran PCR for KCNJ9 HIFI. DpnI digested HIFI pGHE reactions and KCNJ3 and OPRM1 HIFI
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TR1/27Ran gel of KCNJ9 HIFI PCR. Fail- bands but not enough DNA to quantify. Retry PCR
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TR1/27HIFI reaction of OPRM1, KCNJ3
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TR1/28Gel of OPRM1,KCNJ3 HiFi reactions. KCNJ9 DnpI digest for KCNJ9 HIFI.
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CK1/28Did calculations for and picked primers for sequencing of KCNJ3 blunt ended and OPRM1 blunt ended subcloning.
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TR2/3Finished miniprep for OPRM1-1 and OPRM1-2 HIFI
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TR2/4Mini prep of KCNJ9left at RNase step
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CK2/4Analyzed 01/31/20 sequencing results. Blunt ended subcloning did not work for KCNJ3 1-3 and OPRM1 1-6. For blunt ended subcloning in the future, purify PCR products with Purelink PCR purification kit on the bottom shelf of Cabinet A to avoid the unwanted insertion of primer dimersOPRM1-6 is correct in pANT7 if even run out for some reason
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TR2/5Gel concentation OPRM1, KCNJ3
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TR2/6Send OPRM1 and KCNJ3 for sequencing Begining of both confirmed continue with linearization. Save some DNA, also send for confrimation of end