Team:Freiburg/Labjournals/Cloning/July

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Cloning

2015/07/01

Digest of pET_803 (LS)

After sequencing confirmed that we have the correct pET_803 vector (=pET_iGEM with antigen 8 (HSV1) with C-term His-tag), pET_308 was digested with BamHI and HindIII to excise the antigen. Afterwards other antigens can be inserted.

volumeingredient
5µlbuffer
1µlFD BamHI
1µlFD HindIII
10µDNA
33µdH2O
  • 5min at 37°C
  • Dephosphorylation (to avoid religation of the backbone):
volumeingredient
6µAntarctic phosphatase buffer
1µlAntarctic phosphatase
50µDigest
3µdH2O
  • 1h, 37°C
  • analysis on 1%agarose gel failed, nothin was visible, not even the marker. Something seems to be wrong with the gel or buffer,…

Digest of pET_803 (RE)

The digest was repeated, because the first time nothing was visible on the gel.

volumeingredient
5 µl FD green buffer
1 µl BamHI
1 µl HindIII
5 µl DNA
38 µl dH2O
  • excision of antigen 8 with BamHI and HindIII so that other antigens can be inserted in the backbone with C-term. 10xHis-tag
  • 5min at 37°C
  • expected fragment size: ~5.400 bp
  • analyis on 1% agarose gel: again nothing could be seen on the gel –> running buffer will be exchanged for the next runs!

2015/07/02

Digest of pET_803 (RE)

Yesterday's digest of pET_803 had to be repeated again. Gel runs of CellfEx confirmed that the exchange of the running buffer was sufficient to solve yesterday's problems.
Since a lot of backbone of pET_803 is needed for ligation with all the antigens the digest was performed in a larger scale.

volume (1x) volume (5x) ingredient
5 µl 25 µl FD Green buffer
1 µl 5 µl BamHI
1 µl 5 µl HindIII
4 µl 20 µl DNA
39 µl 195 µl dH2O
  • fragment size of the backbone: ~5400 bp
  • analysis on 1% agarose gel:

Gel-ex of pET_803 backbone (RE)

The expected 5400 bp fragment was excised from the agarose-gel and cleaned-up.

  • 803 = Herpes simplex antigen; BamHI and HindIII digested
  • Qiagen kit
  • eluted in 30µl dH2O
  • Nano-Drop:

Miniprep of pET_804 ligation clones(LS)

Ligation of pET_iGEM (digested BamHI + AflII) with antigen 8 (HSV1/ digested with BamHI + AflII) was done two days before. Clones were picked from the clones and inoculated in liquid culture. Miniprep of the liquid cultures was performed to further analyse the plasmid via test-digest.

  • Qiagen kit
  • concentrations:
pET_804 (1)Ligation 1 234,9 ng/µl
pET_804 (2)Ligation 1 200,4 ng/µµl
pET_804 (3)Ligation 1 127,9ng/µl
pET_804 (4)Ligation 1 201,5ng/µl
pET_804 (1)Ligation 2 245,5ng/µl
pET_803 301,1/260,4 ng/µl

Test digest of pET_804 with BamHI and HindIII (LS)

volumeingredient
2µlbuffer
1µlBamHI
1µlHindIII
5µlDNA
11µldH2O
  • 5 min, 37°C
  • analysis on 1% agarose gel
Test digest of different ligation clones, from two different ligations (named L1 and L2).

Digest of aniges 10 (Hepatitis), 11(Tetanus) and 17(HIV) (LS)

The antigenes 10, 11 and 17 were the first to be digested with BamHI and HindIII, because we already have the respective antibodies for further experiments. The antigens were digested out of pJET. Each antigen was digested 3 times, therefore a 10x MasterMix was prepared.

volumeingredient
450µlbuffer
10µlBamHI
10µlHindIII
380µldH2O
  • 5µl DNA per digest (~1µg)
  • 5 min, 37°C
  • expected fragments:
  • antigen 10: 555bp
  • antigen 11: 1363 bp
  • antigen 17: 550bp

Gel-ex of antigen 10, 11 and 17 (LS)

Correct fragments were excised from the agarose-gel and cleaned-up.

  • BamHI and HindIII digested
  • Qiagen kit:
  • yellow buffer turned slightly violet
  • addition of 15µlKAc (3M)
  • nothing changed
  • addition of 50µl KAc (3M)
  • still no change in color
  • proceeded according to protocol nevertheless
  • elution in 20µl dH2O

Ligation of digested pET_803 backbone with digested antigens (LS)

To be able to express different antigens with a His-tag, the digested antigen 10 (Hepatitis), 11 (Tetanus) and 17 (HIV) are ligated in the pET_803 backbone. The pET_803 backbone still has the His-tag while the antigen 8 (HSV1) is excised.

volumeingredient
0,71µlpET_803 backbone
1,28µlantigen 10
2,31µlantigen 11
1,57µlantigen 17
2µlT4 ligase buffer
1µlT4 ligase
up to 20µl dH2O
  • incubation at RT, ~1h
  • ligation control without Insert, instead:H2O

Transformation of Ligation into E.coli Top 10 (LS)

  • 10µl of ligation was used
  • transformation according to protocol
  • plated on LB-Amp plates
  • incubation at 37°, o/n

2015/07/03

Inocculation of 4 clones from each ligation (antigens 10, 11 and 17) (RE)

The ligation worked good. Some colonies visible on the ligation control plate, but far more on the ligation plates.
4 single colonies were picked from each plate. Successful ligation will be verified by test digest after mini-prepping the different clones.

  • inoculated in 5ml culture
  • incubation at 37°, shaking

Digest of pIG15_104 (RE)

  • pIG15_104 = turboYFP with N-term. His and C-term. Spy-tag in the backbone for cell-free expression
  • digest with BamHI and HindIII allows to exchange TurboYFP with the antigens.
volume ingredient
5 µl FD Green buffer
1 µl BamHI
1 µl HindIII
7 µl DNA
36 µl dH2O
  • 5min, 37°C

Digest of antigens 6 (Rubella) and 18 (Treponema) (RE)

volume ingredient
5 µl FD Green buffer
1 µl BamHI
1 µl HindIII
5 µl / 7 µl pIG15_601 / pIG15_1801
38 / 36 µl dH2O
  • both in double amount to get more of the resulting antigen fragment
  • pIG16_601 / _1801 –> antigens 6 / 8 in pJet
  • 37°C, 5 min

Analysis of both digests on 1% agarose gel (RE)

  • pIG15_104: backbone (~3.000bp) excised
  • pIG15_601: nothing (too small? –> digest more DNA next time)
  • pIG15_1801: antigen 18 (~550bp) excised

Gel-Ex (RE)

  • pIG15_104 backbone: 4,9 ng/µl
  • fragment 1801 (antigen 18): 2,8 ng/µl
  • Qiagen-Kit
  • eluted in 20 µl dH2O

Transformation (RE)

  • pIG15_104, pIG15_601 and pIG15_1801
  • into E.coli Top 10

2015/07/04

Miniprep (LS)

  • 6 clones from the pETiGEM_804 ligation (antigen 804 with N-term His-Tag and C-term Spy-Tag)
  • additionally: pIG15_104 (=backbone for cellfree expression), pIG15_601(Rubella Virus) and pIG15_1801 (Treponema pallidum)
  • qiagen kit was used
  • concentrations:
pET_804 (3) Ligation 2139,0
pET_804 (4) Ligation 276,6
pET_804 (5) Ligation 2132,6
pET_804 (6) Ligation 274,6
pET_804 (7) Ligation 2121,7
pET_804 (8) Ligation 2103,7
2x pIG15_104238,8/ 448,6
2x pIG15_601380,3/ 222,7
2x pIG15_1801493,4/ 121,4

Digest of cellfree backbone (pIG15_104) and pIG15_601/1801 (LS)

volumeingredient
5µlFD Green buffer
1µlBamHI
1µlHindIII
5µlDNA
38µldH2O
  • expected fragments:
  • pIG15_104: backbone ~3000bp
  • pIG15_601: insert ~225bp
  • pIG15_1801: insert ~550bp
  • 5 min, 37°C

Test Digest: pET_804, pET_1003, pET_1103 and pET_1703 (LS)

  • pET_804: antigen 8 with N-term His-Tag and C-term Spy-tag
  • pET_1003: antigen 10 with N-term His-tag and C-term Spy-tag
  • pET_1103: antigen 11 with N-term His-tag and C-term Spy-tag
  • pET_1703: antigen 17 with N-term His-tag and C-term Spy-tag
  • MasterMix
volumeingredient
38µlFD green buffer
19µlBamHI
19µlHindIII
5µl eachDNA
209µldH2O
  • 5 min, 37°C
  • expected fragments:
  • pET_804: 390bp + ~5500bp
  • pET_1003: 570bp + ~5500bp
  • pET_1103: 1352bp + ~5500bp
  • pET_1703: 555bp + ~5500bp
Test digest to verify correct insertion of the antigens into the pETiGEM backbone after liagtion. Correct bands are visible for all clones except for the pET_804 clones.

Digest: pETiGEM + fragment 804 (LS)

volumeingredient
5µlFD green buffer
1µlBamHI
1µlAflII(fr 804)/HindIII (pETiGEM)
10µlDNA
33µldH2O
  • 5min, 37°C
Digest of pETiGEM for ligation with different antigens.

Gel-ex: pIG15_104 (cellfree backbone), fragment 601 (Rubella Virus) and fragment 1801 (Treponema pallidum) (JN)

  • concentrations:
pIG15_10441,6
fragment 6018,7
fragment 180111,1

Ligation: pIG15_104 with antigen 6/18 and pETiGEM with antigen 6 (JN)

ingredientvolume
pIG15_104 (=cellfree backbone)1,2µl
antigen 61,4µl
antigen 182,5µl
T4 ligase buffer2µl
T4 ligase1µl
dH2Oup to 20µl
pETiGEM14,4µl
antigen 60,7µl
T4 ligase buffer2µl
T4 ligase1µl
dH2Oup to 20µl
  • incubation for 1h at RT
  • ligation control of pIG15_104 without insert

Gel-ex: pETiGEM (digested) and fragment 804 (digested) (LS)

  • qiagen kit was used
  • concentrations:
pETiGEM (BamHI+AflII)22,5
804 (BamHI+AflII)54,6
pETiGEM (BamHI+HindIII19,5

Ligation: pETiGEM with fragment 804 (LS)

pETiGEM2,22µl
fragment 8040,22µl
T4 ligase buffer2µl
T4 ligase1µl
dH2Oup to 20µl
  • ligation control without insert
  • 1h, RT

Transformation of ligation into E.coli Top 10 (LS)

  • 10µl of ligation
  • transformation according to protocol

2015/07/05

Ligation: antigen 10, 11 and 17 into the cellfree backbone (LS)

antigen 10antigen 11antigen17
backbone1,2µl1,2µl1,2µl
insert0,94µl2,29µl1,5µl
T4 ligase buffer 2µl
T4 ligase 1µl
dH2O up to 20µl
  • 1h, RT

2015/07/06

Miniprep of pIG15_1804/604 and pETiGEM_602/804 clones (JN)

concentrations:

pIG15_1804 (1)208,3
pIG15_1804 (2)95,1
pIG15_1804 (3)115,6
pIG15_604 (1)97,1
pIG15_604 (2)469
pIG15_604 (3)157,2
pETiGEM_602 (1)160,1
pETiGEM_602 (2)166,9
pETiGEM_602 (3)148,6
pETiGEM_804 (1)162,4
pETiGEM_804 (2)168,6
pETiGEM_804 (3)157,9

Test-Digest of all clones above (JN)

volumeingredient
2µlbuffer
1µlRE1
1µlRE2
5µlDNA
11µldH2O
  • for pIG15_604: EcoRI and HindIII
  • for pET_602: PstI and HindIII
  • for pIG15_1804/pET_iGEM_804: BamHI and HindIII
Test digest. Expected fragments will follow soon…

2015/07/07

Dephosphorylation of pET_iGEM BamHI and AlfII digested (RE)

  • 1 µl Antartic phosphatase
  • 2 µl Antartic phosphatase buffer
  • added to the digest product
  • 1 h at 37°C
  • heat inactiation 5 min at 70°C

Ligation (RE)

  • pET_iGEM + fragment 804 (BamHI and AflII digested)
804 0.31 µl
pET_iGEM 2.17 µl
T4 ligase 1 µl
T4 ligase buffer 5 µl
dH2O 41.52 µl

Mini-Prep (RE)

  • pIG15_1004, pIG15_1104 and pIG15_1704 (His- and Spy-tagged constructs for cellfree expression)
  • Qiagen-Kit
  • eluted in 30 µl dH2O
  • Nano-Drop:
pIG15_1004-1 261.0 ng/µl
pIG15_1004-2 490.3 ng/µl
pIG15_1004-3 414.8 ng/µl
pIG15_1104-1 860 ng/µl
pIG15_1104-2 120.6 ng/µl
pIG15_1104-3 174.6 ng/µl
pIG15_1704-1 505.2 ng/µl
pIG15_1704-2 90.5 ng/µl
pIG15_1704-3 895.2 ng/µl

Test digest (RE)

  • pIG15_1004, pIG15_1104 and pIG15_1704
  • BamHI and HindIII digest
  • expected fragment sizes: backbone of all constructs ~3000 bp; insert 1004 ~600 bp; insert 1104 ~1300 bp; insert 1704 ~550 bp
1x 10x
BamHI 1 µl 10 µl
HindIII 1 µl 10 µl
FD Green Buffer 5 µl 50 µl
DNA 5 µl -
dH2O 11 µl 110 µl
Test digest for pIG1004, pIG1104 and pIG1704, which are the different antigens with a C-term Spy-tag. Expected fragments for antigen 10: ~600bp, for antigen 11: ~1300bp and for antigen 1704: ~550bp.

PCR for fragment 804 (RE)

  • Herpes simplex antigen with His- and Spy-tag
Taq pol 1.5 µl
Taq buffer 30 µl
dNTPs 3 µl
oIG15_803f 3 µl
oIG15_804r 3 µl
pIG15_801 1 µl
dH2o 58.5 µl

cycling condition as for Taq-PCR from 06/30/2015

PCR clean-up (RE)

  • Qiagen-Kit
  • eluted in 40 µl dH2O
  • NanoDrop: 207.9 ng/µl

AflII digest of fragment 804 (PCR product) (RE)

  • 2 µl AflII
  • 5 µl CutSmart
  • 10 µl DNA
  • 33 µl dH2O
  • 2 h at 37°C –> clean-up as above

2015/07/08

Digest: PCR product 804 + pETiGEM with Acc65I (LS)

volumeingredient
5µlbuffer 3.1
1µlAcc65I
10µlpETiGEM
30µlPCR product 804
10µl PCR product 804 already digested with AflII
up to 50 µldH2O
  • 1h, 37°C
  • accidently did this digest, Acc65I digest was already done yesterday
  • all constructs except pETiGEM are digested twice with Acc65I

PCR for 804 (LS)

  • same as on 21.6.2015
  • clean-up with qiagen PCR Clean-up kit

Digest of PCr product 804 with Acc65I/DpnI (LS)

volumeingredient
5µlbuffer
1µlAcc6I
1µlDpnI
20µlDNA
up to 50µldH2O
  • 1h, 37°C
  • clean-up with qiagen kit

Digest of pETiGEM with Acc65I (LS)

volumeingredient
5µlbuffer
1µlAcc65I
10µlDNA
up to 50µldH2O
  • 1h, 37°C
  • clean-up with qiagen kit

Digest of pETiGEM and pCR product 804 with AflII (BspI) (LS)

volumeingredient
5µlFD green buffer
1µlBspI
20µlDNA
up to 20µldH2O
  • 5min, 37°C
  • clean-up with qiagen kit

Ligation: pETiGEM + 804 (LS)

volumeingredient
2µlT4 ligase buffer
1µlT4 ligase
4µlpETiGEM
1µlfragment 804
up to 20µldH2O
  • ligation control without insert
  • 1h at RT

Dephosphorylation of digested pETiGEM (LS)

volumeingredient
2µAntarctic phosphatase buffer
1µlAntarctic phosphatase
10µlpEtiGEM
up to 20µldH2O
  • 1h, 37°C
  • clean-up with qiagen kit

2015/07/09

Transformation (RE)

  • Ligation product of pET_iGEM + 804 (+/- dephosphorylation of the backbone)
  • Ligation controls for both backbones (+/- dephosphorylation)

Inocculation (RE)

  • 4 x 5 ml liqid culture (LB amp)
  • pET_iGEM in E. coli TOP10

Dephosphorylation (JN)

  • dephosphorylation of the cell-free backbone pIG15_104 that was digested with BamHI and HindIII (15/07/03, RE, 4,9ng/µl) for upcoming ligation
volumeingredient
5µlAntarctic phosphatase buffer
1µlAntarctic phosphatase
16µlDigest
28µldH2O
  • incubation at 37°C for 1h
  • heat inactivation for usage without plasmid purification: 70°C for 5min

Ligation (JN)

  • pIG15_104 with concentration of 1.6ng/µl (due to dephosphorylation)
antigen 10antigen 11antigen17control
backbone12,5µl12,5µl12,5µl12,5
insert1,13µl2,43µl1,5µl -
T4 ligase buffer 2µl
T4 ligase 1µl
dH2O up to 20µl
  • 1h at RT
  • trafo according to protocol

2015/07/10

Inoculation (JN)

  • pET_804 (construct of Herpes with Spy Tag) with and without dephosphorylation
  • 3x5ml liquid cultures each

Mini-Prep (JN)

  • pET_iGEM
  • Qiagen kit
  • eluted in 30µl H2O
pET_iGEM1 104.9ng/µl
pET_iGEM2 109.0ng/µl

Mini-Prep (JN)

  • pET_804 (construct of Herpes with Spy Tag) with and without dephosphorylation
  • Qiagen kit
  • eluted in 30µl H2O
pET_804 with dephos. 1 245.5ng/µl
pET_804 with dephos. 2 350.3ng/µl
pET_804 with dephos. 3 207.1ng/µl
pET_804 without dephos. 1 248.2ng/µl
pET_804 without dephos. 2 298.8ng/µl
pET_804 without dephos. 3 372.6ng/µl

Inoculation (JN)

  • pIG15_1004/1104/1704 (cell-free expression constructs with His and Spy Tag)
  • 3x5ml each

2015/07/11

Mini-Prep (RE)

  • pIG15_1004, pIG15_1104 and pIG15_1704 (3 clones each)
  • Qiagen-Kit
  • eluted in 30 µl dH2O
  • NanoDrop:
pIG15_1004 - 1 302.7 ng/µl
pIG15_1004 - 2 247.7
pIG15_1004 - 3 272.7
pIG15_1104 - 1 330.3
pIG15_1104 - 2 313.0
pIG15_1104 - 3 336.6
pIG15_1704 - 1 297.0
pIG15_1704 - 2 334.9
pIG15_1704 - 3 285.0

2015/07/12

Test-Digest (JN)

  • Test-Digest of pET_804 and pIG15_1004/1104/1704
pET_804 Master Mix (x7) pIG15 Master Mix (x10)
DNA 5µl - DNA 5µl -
Xba 1µl 7µl EcoRI 1µl 10µl
HindIII 1µl 7µl HindIII 1µl 10µl
CutSmart 2µl 14µl CutSmart 2µl 20µl
H2O 11µl 77µl H2O 11µl 110µl
  • in total 20µl
  • incubation 1h, 37°C
  • 20min 80°C heat inactivation
  • !! mistakenly the digest Master Mix was pipetted into pET_804+desphosphrylation 1: instant heat inactivation and purification with PCR kit (Qiagen) was performed and elution was perforemd in 25µl H2O; concentration: 121.5ng/µl !!

Ligation (RE)

  • antigens 6 and 18 into pET_803 (BamHI and HindIII digested)
6 (Rubella) 18 (Treponema) Ligation control
backbone 0.71 µl 0.71 µl 0.71 µl
insert 0.71 µl 1.26 µl -
T4 Ligase 1 µl 1 µl 1 µl
T4 Ligase buffer 2 µl 2 µl 2 µl
dH2O 15.58 µl 15.03 µl 16.29 µl

–> 1 h at RT

Transformation (RE)

  • Ligation products pET_603 and pET_1803
  • 5 µl of each sample were used for transformation

Taq PCR for fragment 804 (RE)

  • Herpes simplex antigen with His- and Spy-tag
1x 9x
pIG15_801 1 µl 9 µl
oIG15_803f 1.5 µl 13.5 µl
pIG15_804r 1.5 µl 13.5 µl
Dream Taq 0.6 µl 5.4 µl
Taq Buffer 5 µl 45 µl
dNTPs 2.5 µl 22.5 µl
dH2O 37.9 µl 341.1 µl

Cycling conditions:

95°C 30 s
95°C 20 s
55°C 50 s
68°C 60 s
68°C 300 s
4°C infinite

PCR clean-up (RE)

  • fragment 804
  • 4 of 8 samples were cleaned up without checking the PCR result on an agarose gel
  • Qiagen-Kit
  • eluted in 2×30 µl dH2O
  • NanoDrop: 104.7 and 86.7 ng/µl

DpnI digest of the purified PCR product (804) (RE)

  • 1 µl DpnI
  • 2 µl CutSmart buffer
  • directly added to the purified PCR product
  • incubated at 37°C o/n

Gelelectrophoresis and Gel-Ex(RE)

  • the other 4 PCR samples (804) were analyzed by gelelectrophoresis and extracted from the gel
  • Qiagen-Kit
  • eluted in 2×20 µl dH2O

AflII digest (JN)

  • fragment 804 (gel extracted)
  • pET_iGEM
804 pET_iGEM
DNA 5µl 10µl
AflII (BspTI) 1µl 1µl
FD buffer 5µl 5µl
H2O 39µl 34µl
  • in total 50µl
  • incubation for 5min, 37°C

Purification of the digest products (JN)

  • Qiagen PCR purification kit
  • eluted in 25µl
concentration
804(1)
804(2)
pET_iGEM(1)
pET_iGEM(2)

Acc65I digest (JN)

  • purified fragment 804 and pET_iGEM
804 pET_iGEM
DNA 15µl 15µl
Acc65I 1µl 1µl
3.1 buffer 5µl 5µl
H2O 29µl 29µl
  • in total 50µl
  • incubation for 1h, 37°C

Purification of the digest products (JN)

  • Qiagen PCR purification kit
  • eluted in 20µl

| | concentration |

804(1) 32.2ng/µl
804(2) 18.4ng/µl
pET_iGEM(1) 6.8ng/µl
pET_iGEM(2) 7.7ng/µl

Ligation (RE)

  • pET_iGEM (not dephosphorylated) + fragment 804
  • both AflII and Acc65I digested
pET_iGEM 1.56 µl
fragment 804 1.88 µl
T4 Ligase 1 µl
T4 Ligase buffer 2 µl
dH20 13.56 µl

–> incubated 1 h at RT

Transformation (RE)

  • Ligation product (pET_iGEM (not dephosphorylated) + fragment 804)
  • 5 µl used

Dephosphorylation (RE)

  • one sample (7.7 g/µl) of pET_iGEM (AflII and Acc65I digested)
  • 1 µl Antarctic phosphatase
  • 2 µl Antarctic phosphatase buffer
  • directly added to the purified digest product
  • incubated 1 h at 37°C

Ligation (RE)

  • pET_iGEM (dephosphorylated) + fragment 804
  • both AflII and Acc65I digested
pET_iGEM 1.56 µl
fragment 804 1.88 µl
T4 Ligase 1 µl
T4 Ligase buffer 2 µl
dH20 13.56 µl

–> incubated o/n at 16°C

2015/07/15

Transformation (RE)

  • ligation product of pET_iGEM (dephosphorylated) + fragment 804

Clean-up of DpnI-digest of fragment 804 (RE)

  • Qiagen-Kit (PCR clean-up)
  • eluted in 2×20 µl
  • NanoDrop: 41.1 and 53.3 ng/µl

Dephosphorylation (RE)

  • pET_iGEM (AflII and Acc65I digested)
  • 2nd tube
  • exactly as for the first tube yesterday

BspTI (AflII) digest (RE)

  • DpnI digested fragment 804
  • 18 µl DNA (the whole tube)
  • 5 µl FD Grenn Buffer
  • 1 µl BspTI
  • 26 µl dH2O
  • incubated for 5 min at 37°C

Clean-up of the BspTI digest (RE)

  • as for DpnI digest before
  • both samples were applied to one column and eluted in 30 µl dH2O
  • NanoDrop: 29.7 ng/µl

Acc65I digest (RE)

  • DpnI + AflII digested fragment 804
  • 28 µl DNA (the whole tube)
  • 1 µl Acc65I
  • 5 µl NEB Buffer 3.1
  • 16 µl dH2O
  • incubated for 1h at 37°C

Gelelectrophresis and gel extraction (RE)

  • fragment 804 digested with DpnI, AflII and Acc65I
  • fragment of ~500 bp was extracted from the gel –> Herpes simplex antigen with His- and Spy-tag for insertion into pET_iGEM
  • Qiagen-Kit
  • eluted in 15 µl dH2O
  • NanoDrop: 11.7 ng/µl

Ligation (RE)

  • 1.56 µl pET_iGEM (AflII and Acc65I digested, dephosphorylated)
  • 1.16 µl fragment 804 (DpnI, AflII and Acc65I digested)
  • 1 µl T4 Ligase
  • 2 µl T4 Ligase buffer
  • 15.28 µl dH2O

Transformation (RE)

  • 5 µl of the ligation product into E. coli TOP10

Test digest (RE)

  • pET_603 and pET_1803 (07/12)
  • 2 µl FD Green buffer
  • 1 µl HindIII
  • 1 µl BamHI
  • 1 µl (1803) or 2 µl (603) DNA
  • 16 µl (1803) or 15 µl (603) dH2O
  • incubated for 5 min at 37°C

analysis on 1 % agarose gel:

clones 2 and 4 of both constructs were chosen for sequencing (pET_1803: both positive! pET_603: something slightly longer was inserted)

Test digest for pET_804 (LS)

  • Ligation (with/without dephosphorylated backbone)
  • 5 µl DNA
  • 2 µl FD Green buffer
  • 1 µl BamHI
  • 1 µl HindIII
  • 41 µl dH2O (accidently)
  • incubated for 5 min at 37°C
Test digest of ligation clones for pETiGEM_804 (Antigen 8 with N-Term His-tag and C-term Spy-tag. The four clones on the left were ligated into the dephosphorylated backbone, the four clones at the right into the pETiGEM that was not dephosphorylated. The white arrow idicates a hardly visible band at the right size which could be the correct insert (~400bp).

Repetition of test digest for pET_804 (RE)

  • Ligation (with/without dephosphorylated backbone)
  • 5 µl DNA
  • 2 µl FD Green buffer
  • 1 µl BamHI
  • 1 µl HindIII
  • 11 µl dH2O
  • incubated for 5 min at 37°C
Test digest of ligation clones for pETiGEM_804 (Antigen 8 with N-Term His-tag and C-term Spy-tag. The four clones on the right were ligated into the dephosphorylated backbone, the four clones at the left into the pETiGEM that was not dephosphorylated. The white arrow idicates a hardly visible band at the right size which could be the correct insert (~400bp).

2015/07/17

Test digest for pET_804 (with DpnI digest after PCR) (RE)

  • 5 µl DNA
  • 1 µl HindIII-HF
  • 1 µl PstI-HF
  • 2 µl CutSmart
  • 11 ml dH2O
  • incubated for 1h at 37°C

Digest of pIG15_105 (RE)

  • cell-free expression backbone with His-tag - tYFP - Spy-tag
  • digest with BamHI and HindIII to excise tYFP –> insert antigens instead
  • 5 µl DNA
  • 1 µl BamHI
  • 1 µl HindIII
  • 5 µl FD Green Buffer
  • 38 µl dH2O
  • incubated for 5 min at 37°C

analysis on 1 % agarose gel:

- Test digest of pET_804 (with DpnI template digest after PCR): expected fragment sizes ~1700 and ~4100 bp –> clone 1 and 3 will be sequenced
- Digest of pIG15_105: expected fragment sizes ~700 and ~3200 bp –> backbone (3200 bp fragment) will be extracted from the gel





















Gel extraction of pIG15_105 backbone (RE)

  • Qiagen-Kit
  • eluted in 30 µl dH2O
  • NanoDrop: 8.5 ng/µl

Dephosphorylation (RE)

  • pIG15_105 (BamHI and HindIII digested backbone)
  • 1 µl Antarctic phosphatase
  • 3 µl Antarctic phosphatase buffer
  • directly added to the digest product after gel extraction
  • incubated for 1 h at 37°C

Transformation of pIG15_105 (RE)

Ligation pIG15_105 + antigen 8,10, 11 and 17 (LS)

antigens8101117control
antigen fragment (BamHI/HindII)0,619,381,790,83-
pIG15_105 (BamHi/HindIII+Dephospho 5,88
dH2O10,511,749,3311,1212
T4 DNA ligase buffer 2
T4 DNA Ligase 1

2015/07/18

Transformation: Ligation into T10 and pIG15_601; 801; 1001; 1101 and 1701 into T10 (LS)

Transformation of pJet with antigen fragments (pIG15_X01) because more digested antigen fragment was needed.

  • 10µl of ligation
  • 1µl of plasmids (pIG15_X01)
  • transformation accordinh to protocol
  • Ligation on LB-Cml plates
  • pIG15_X01 in LB-Amp liquid culture

Inoculation of 2x 5ml LB-Cml with pIG15_105 (=cell free backbone with Halo-tag) (LS)

To be able to perform additional ligations with this backboen, more template was needed. Therefore liquid cultures of the respective E.colis were incubated at 37° for several hours.

Miniprep: pIg15_X01 (LS)

  • qiagen kit
  • concentration:
pIG15_601393,7
pIG15_80181,2
pIG15_1001304,0
pIG15_1101350,4
pIG15_1701355,2

Digest: pIG15_X01 with BamHI and HindIII (LS)

To excise the antigen fragments out of pJet, the plasmids were digested with BamHI and HindIII.

ingredientvolume
DNA10µl
FD Green buffer5µl
BamHI1µl
HindIIIµl
dH2O up to 50 µl
  • different for pIG15_801: since concentration of the plasmid was really low, 20µl of DNA was used for the digest
  • 5 min, 37°C
  • analysis on 1% agarose gel
Digest of antigen fragments out of pJET_X01. White arrows indicate the correct fragments.
  • gel did look a bit strange, most lanes showed several bands, where only two bands were expected: One for the pJet backbone (~3000bp) and one for the excised antigen fragment.
  • expected band lenghts for the antigen fragments:
6225
8390
10570
111353
17547
  • only antigen 6, 10 and 17 showed correct bands and were excised from the gel

Gel-ex: antigen fragments (LS)

  • gel-ex with qiagen
  • elution in 30µl dH2O
  • concentrations:
antigen fragment 6 8,8
antigen fragment 10 11,9
antigen fragment 17 18,2

Miniprep: pIG15_105 (=cell free backbone with Halo-tag) LS

  • 2x5ml were pelleted
  • qiagen kit, elution in 50µl
  • concentration: 292,2ng/µl

2015/07/20

Digest: pIG15_105 with BamHI and HindIII (LS)

Since there were no clones on the ligation plates, the ligation obviously didn't work. To repeat this ligation the backbone pIG15_105 is digested with BamHI and HindIII.

ingredientvolume
pIG15_1025µl
BamHI1µl
HindIII1µl
FD Green buffer 5µl
dH2Oup to 50µl
  • 5min, 37°C
  • 5 min, 80 °C for heat inactivation

Dephosphorylation of digested pIG15_105 (LS)

To prevent the backbone from re-ligating with itself, the digested backbone is dephosphorylated.

ingredientvolume
digested pIG15_10550µl
Antarctic phosphatase buffer 6µl
Antarctic phosphatase1µl
dH2O3µl
  • 1h, 37°C
  • analysis on 1% agarose gel
Digested (BamHI and HindIII) and dephosphorylated pIG15_105. Upper band is the backbone (~3000bp) and lower band the excised tYFP.

Gel-ex: pIG15_105 (digested and dephosphorylated) (LS)

  • excision of correct bands
  • qiagen kit
  • concentration: 8,8ng/µl

Ligation: Antigen fragments into pIG15_105 (LS)

antigen fragment6018011000111011701Control
volume1,361,112,102,551,37-
pIG15_105 6,17
dH2O9,479,728,738,289,4612
T4 DNA Ligase buffer 2
T4 DNA Ligase 1
  • 1h, RT

Transformation into T10: Ligation + control and pET22b+ (LS)

Since the last ligation didn't work, we want to test the transformation efficiency of the new competent cells. The same new competent cells were used for the last ligation, so maybe it's not a problem of ligation, but of the cells. Therefore pEt22b+ is transformed as a control.

  • 10µl of ligation/control were used
  • 1µl of pEt22b+
  • transformation according to protocol
  • plated on LB-Cml/ LB-Amp respectivly

Inoculation and Platin (JN)

  • plating of pIG15_601/801/1001/1101/1701/1801 (blunt-end cloned pJET with the IDT antigens) from glycerol stocks on Amp plates o/n
  • inoculation of the transformed E.coli with the plasmids mentioned above in 3x5ml LB+Amp o/n

2015/07/21

Inocculation (RE)

  • pIG15_605
  • pIG15_805
  • pIG15_1005
  • pIG15_1105
  • pIG15_1705
  • 3 colonies per construct were picked
  • in 5 ml liquid LB +cml

Mini-Prep (RE)

  • pIG15_601, pIG15_801, pIG15_1001, pIG15_1101, pIG15_1701 and pIG15_1801
  • Qiagen-kit
  • 3 preps per construct –> elutions pooled into one tube per construct –> 90 µl dH2O in total
  • NanoDrop:
pIG15_601 293.2 ng/µl
pIG15_801 592.7 ng/µl
pIG15_1001 523.9 ng/µl
pIG15_1101 548.6 ng/µl
pIG15_1701 674.5 ng/µl
pIG15_1801 720.8 ng/µl

Digest to excise antigens from pJet (RE)

  • pIG15_601, pIG15_801, pIG15_1001, pIG15_1101, pIG15_1701 and pIG15_1801
  • 5 µl DNA
  • 1 µl BamHI
  • 1 µl HindIII
  • 5 µl FD Green Buffer
  • 38 µl dH2O
  • two samples per antigen

analysis on 1% agarose gel:

Repetition of the digest above (RE)

  • same as before, but:
  • NEB enzymes and CutSmart buffer were used

analysis on 1% agarose gel:

2015/07/22

Test digest of pET_804 (RE)

  • TD was repeated, because the former TD (07/16) seemed to be positive, but the sequencing results were negative
  • the constructs were digested with PstI-HF/HindIII-HF and PstI-HF/BamHI to verify the insert by length differences between one of the fragments: without insert the smaller fragment should be about 1350 bp in all cases, but if the insertion was successful, the PstI/BamHI digest should result in a ~1750 bp fragment
  • 5 µl DNA
  • 1 µl PstI-HF
  • 1 µl BamHI/HindIII
  • 2 µl CutSmart
  • 11 µl dH2O
  • incubation at 37°C for 1 h

analysis on 1% agarose gel:

Expected fragement sizes for successful insertion of fragment 804: PstI/HindIII - ~1350 and ~4100 bp; PstI/BamHI - ~1750 and 4100 bp; –> insert could not be verified

Test digest of constructs for cell-free expression (RE)

  • pIG15_605 –> 225 bp
  • pIG15_805 –> 390 bp
  • pIG15_1005 –> 570 bp
  • pIG15_1105 –> 1350 bp
  • pIG15_1705 –> 540 bp
  • cell-free expression backbone for Halo-tagged antigens
  • 5 µl DNA
  • 1 µl BamHI
  • 1 µl HindIII
  • 2 µl FD Green Buffer
  • 11 µl dH2O
  • incubation at 37°C for 5 min

analysis on 1% agarose gel:

Expected fragment sizes of the antigens are mentioned above; backbone: ~3000 bp
  • pIG15_605 - 3
  • pIG15_805 - 1 and - 3
  • pIG15_1005 - 2 and - 3
  • pIG15_1105 - 2 and - 3
  • pIG15_1705 - 1 and - 2
  • –> will be sequenced!
  • sequencing is irrelevant, because a systematic mistake in the backbone was found which results in a framshift after inserting an antigen every time

2015/07/23

Mini-Prep of pJet_804 (JN)

  • Qiagen kit
  • eluted in 30µl dH2O
  • concentrations
pJet_804 (1)210.1ng/µl
pJet_804 (2)95.6ng/µl
pJet_804 (3)208.0ng/µl
pJet_804 (4)176.9ng/µl

Test-digest of pJet_804 1-4 (JN)

  • verification of correct insertion of the 804 fragment into linearized pJet backbone
ingredientamount
DNA5µl
BamHI1µl
HindIII1µl
FD buffer2µl
H2O11µl
  • total of 20µl
  • incubation for 5min at 37°C
  • analysis on 1% agarose gel
expected fragment sizes are ~400bp for the insert and ~3000bp for the backbone –> verification not successful

Gel-ex of fragments 6/8/10/11/17/18 (JN)

  • the BamHI and HindIII digested antigen fragments from 2015/07/22 were extracted out of the gel
  • Qiagen kit
  • eluted in 20µl dH2O
  • concentrations
fragment 612.7ng/µl
fragment 829.1ng/µl
fragment 1012.9ng/µl
fragment 1158.5ng/µl
fragment 1710.8ng/µl
fragment 1811.2ng/µl

Ligation of pJet and 804 fragment (JN)

  • linearized pJet backbone and the 804 PCR fragment were ligated
ingredientamount
pJet backbone0.5µl
804 fragment1µl
ligase1µl
ligase buffer2µl
H2O15.5µl
  • total amount of 20µl
  • 5min at RT
  • transformation according to protocol
  • plated on Amp plates o/n

2015/07/24

Picked 4 clones from ligation: pJet_804 (LS)

  • incubation in LB-Amp
  • 37°C, shaking

Ligation: pET_803 (digested with BamHI and HindIII) and antigen 6 (digested with BamHI and HindIII) (LS)

volumeingredient
2µlT4 ligase buffer
0,71µlbackbone
0,57µlinsert
1µlT4 DNA Ligase
15,72µldH2O
  • 1h, RT
  • transformation into E.coli T10
  • platedon LB-Amp

Miniprep: pJet_804 clones (LS)

  • qiagen kit
  • concentratons:
pJet_804 (1)45,1
pJet_804 (2)51,4
pJet_804 (3)48,1
pJet_804 (4)55,8

Test digest: pJet_804 with BamHI and HindIII (LS)

volumeingredient
2µlFD Green buffer
1µlBamHI
1µlHindIII
5µlDNA
11µldH2O
  • analysis on 1% agarose gel
  • no correct insertion of fragment 804
4 clones of pJet_804 were tested. None of the clones has the correct fragment 804 inserted, as there are no bands visible at ~500bp

2015/07/25

Picked clones from pET_603 ligation clones(LS)

  • incubation in 5ml LB-Amp
  • 37°C

Miniprep: biobricks for interlab study (LS)

  • qiagen kit
  • concentrations in 40µl:
Bba_I 13505111,9
Bba_k 82300876,3
Bba_k 823013109,0
Bba_k 833005176,7
  • before miniprep: 50µl of each culture on LB-Cml plates

Miniprep: pET_603 ligation clones (LS)

  • 4 clones
  • qiagen kit
  • concentrations:
pET_603 (1)146,5
pET_603 (2)151,2
pET_603 (3)161,5
pET_603 (4)186,0

pJet blunt end ligation with fragment 804 (RE/LS)

  • Fragment 804 was obtained by DreamTaq PCR, which results in a one base overhang making blunt-end ligation impossible.
  • Therefore, the blunting enzyme was used according to the referring protocol before ligation.
  • The ligation product was transformed into E. coli TOP10

2015/07/26

Test digest of pET_603 clones with XbaI and HindIII (LS/JE)

volumeingredient
2µlbuffer
1µlXbaI
1µlHindIII
5µlDNA
11µldH2O
  • incubation at 37°C for 1,5 h
  • analysis on 1% agarose gel:
All clones showed really faint bands at the correct size (~400bp). On the right side of the gel, we tested some 1kb ladders, that we found in the freezer.

Mini-prep of pJet+804 (RE)

  • Qiagen-kit
  • eluted in 30 µl dH2O
  • NanoDrop:
pJet+804 - 1 322.2 ng/µl
pJet+804 - 2 194.1 ng/µl
pJet+804 - 3 227.8 ng/µl
pJet+804 - 4 249.1 ng/µl

Test digest of pJet+804 (RE)

  • 5 µl DNA
  • 1 µl BamHI
  • 1 µl HindIII
  • 2 µl FD Green Buffer
  • 11 µl dH20
  • expected fragment sizes: ~400 bp and ~3000 bp

1. Digest of pJet+804 (4) (RE)

  • 1 µl BspTI (AflII)
  • 5 µl FD Buffer
  • 4 µl DNA
  • 40 µl dH2O
  • incubated for 5 min at 37°C
  • cleaned up (Qiagen PCR clean-up kit); eluted in 20 µl dH2O

2. Digest of pJet+804 (RE)

  • 1 µl Acc65I
  • 5 µl NEB Buffer 3.1
  • 20 µl Clean-up product
  • 24 µl dH2O
  • incubated for 1 h at 37°C

analysis on 1 % agarose gel:

pJet_804 digested with Acc65I and AflII should result in a fragment of ~500 bp length, but did not work as expected

2015/07/27

Repetition of yesterday's test digest of pJet_804 (RE)

  • less enzyme was used, because yesterday it seemed as if the enzymes destroied the whole plasmid more or less
  • 5 µl DNA
  • 2 µl FD Green Buffer
  • 0.5 µl BamHI
  • 0.5 µl HindIII
  • 12 µl dH2O
  • incubated for 5 min at 37°C and directly chilled on ice

analysis on 1% agarose gel:

Test digest of pJet_804 with BamHI and HindIII; expected fragment sizes ~500 bp and ~3,000 bp
  • Looks like the enzymes were too active again, but clones 2, 3 and 4 are being sequenced anyways.
  • edit 2015/07/28: sequencing result was positive for clone 3!

Inoculation (JN)

  • inoculation of pJet_804-2/3/4
  • re-trafo was split into 2 –> 250µl of each trafo were inoculated in 5ml LB-Amp o/n

2015/07/28

Mini-Prep of pJet_804-3 (JN)

  • pJet_804-3 was the only colony with a positive sequencing result. Therefore, the two liquid cultures of this clone were prepped and the other four were discarded.
  • Qiagen kit
  • eluted in 30µl dH2O
  • concentrations
pJet_804-3 (1)469.2ng/µl
pJet_804-3 (2)471.1ng/µl

Digest of pJet_804 with Acc65I(JN)

ingredient amount [µl]
DNA 5
Acc65I 1
buffer 3.1 5
H2O 39
  • 1h at 37°C
  • afterwards clean-up with PCR purification kit (Qiagen)
  • eluted in 40µl H2O
  • concentrations
pJet_804 - 1 13.5ng/µl
pJet_804 - 2 25.3ng/µl

Digest of pJet_804_Acc65I_digested with AflII/Digest of pJet_804 with AflII and HindIII (JN)

ingredient pJet_804 pJet_804_Acc65I_digested undigested
DNA 5µl 36µl 3µl
FD buffer 5µl 5µl 5µl
AflII 1µl 1µl -
HindIII 1µl - -
dH2O 38µl 8µl 42µl
  • 5min at 37°C
  • analysis on 1% agarose gel

  • 804 insert (cut with Acc65I and AflII) was cut out; backbone was wrongly cut due to inverted insertion into the pJet before

2015/07/29

Repetition of ligation of fragment 804 and pET-iGEM (LS)

PCR for amplification of GST (RE)

  • 0.3 µl Template (1026_pGEX1H6_F)
  • 3 µl of each primer (oIG15_SpyC_fwd and oIG15_SpyC_rev)
  • 12 µl High GC phusion buffer
  • 1.2 µl dNTPs
  • 1.8 µl DMSO
  • 0.6 µl phusion polymerase
  • 38.1 µl dH2O

3 samples à 20 µl

cycling (20 cycles):

  • 30 s at 92°C
  • 20 s at 92°C
  • 20 s at 60°C
  • 60 s at 72°C
  • 300 s at 72°C

After analysis on 1 % agarose gel, the fragment of ~700 bp was extracted with the Qiagen-Kit. Elution was done in 30 µl dH2O ( x ng/µl)

PCR for amplification of fragment 804 (for Gibson in pET) (RE)

  • 0.6 µl Template (pIG15_801)
  • 3 µl of each primer (oIG15_807f and oIG15_808r)
  • 12 µl High GC phusion buffer
  • 1.2 µl dNTPs
  • 1.8 µl DMSO
  • 0.6 µl phusion polymerase
  • 37.8 µl dH2O

3 samples à 20 µl

cycling (20 cycles):

  • 30 s at 92°C
  • 20 s at 92°C
  • 20 s at 60°C
  • 60 s at 72°C
  • 300 s at 72°C

PCR for amplification of fragment 804 (for Gibson in pIG15 with Spy-tag –> w/o frameshift) (RE)

  • 0.3 µl Template (pIG15_104)
  • 3 µl of each primer (oIG15_805f and oIG15_806r)
  • 12 µl High GC phusion buffer
  • 1.2 µl dNTPs
  • 1.8 µl DMSO
  • 0.6 µl phusion polymerase
  • 38.1 µl dH2O

3 samples à 20 µl

cycling (20 cycles):

  • 30 s at 92°C
  • 20 s at 92°C
  • 20 s at 60°C
  • 60 s at 72°C
  • 300 s at 72°C

comment:

  • PCR for fragment 804 with overhangs for Gibson assembly in pET did not work and will be repeated with less template
  • PCR – “ – in pIG15: the wrong template was used, it has to be pJet_804!

Repetition of the PCRs for fragment 804 (RE)

  • PCRs were performed as before with the following changes:
  • pET: only 0.4 µl template were used (instead of 0.6 µl)
  • pIG: pJet_804 was used as template (instead of pIG15_104)

analysis on 1 % agarose gel: successful amplification of the 500 bp fragment in both cases –> extracted

Transformation of pET_SpyC into E. coli TOP10 (LS)

Digest of the biobrick plasmids for the interlab study (LS)

Gel-Ex of the biobrick parts for interlab study and the 804 PCR products (LS)

Ligation of GFP into the different promoter backbones (RE)

  • Interlab study
  • GFP drived from Bba_I13504
Backbone Bba_K823005 Bba_K823008 Bba_K823013
BB amount [µl] 1.25 1.25 0.91
GFP [µl] 2.28 2.28 2.28
T4 Ligase [µl] 1 1 1
T4 Ligase buffer [µl] 2 2 2
dH2O [µl] 16.53 16.53 14.19

Transformation of the ligation products into E. coli TOP10 (RE)

  • 5 µl ligation product used
  • plated on LB_cml

Gibson assembly (RE)

  • pIG15_104 (BB, Acc65I and AflII digested; 1.41 µl) + fragment 804 (Insert; 0.48 µl) + 3.11 µl dH2O –> pIG15_804 (w/o frameshift)
  • pET_iGEM (BB, Acc65I and AflII digested; 4.78 µl) + fragment 804 (Insert; 0.22 µl) –> pET_804
  • directly transformed into E.coli TOP10 (5 µl of the Gibson mix)
  • pIG15_804 was plated on LB_cml
  • pET_804 was plated on LB_amp

2015/07/30

Mini-prep (RE)

  • pET_SpyC (2 cultures)
  • Qiagen-kit
  • eluted in 30 µl
  • NanoDrop

Digest of pET_SpyC (RE)

  • open the backbone to insert GST
  • 1 µl HindIII
  • 1 µl XhoI
  • 7 µl DNA (~1 µg)
  • 5 µl CutSmart
  • 36 µl dH2O
  • incubated for 1 h at 37°C

analysis on 1% agarose gel and extraction of the 5,400 bp fragment:

  • Qiagen-kit
  • eluted in 30 µl dH2O
  • NanoDrop: 12.4 and 12.7 ng/µl

Gibson assembly of pET_SpyC and GST PCR product (LS)

Ligation of pET_iGEM and antigens 8, 10, 11, 15 and 17 (JN)

ingredient antigen 8 10 11 15 17
pET_iGEM bb 2.56 2.56 2.56 2.56 2.56
insert 0.47 1.25 0.65 0.66 1.36
T4 buffer 2 2 2 2 2
T4 ligase 1 1 1 1 1
H2O 13.97 13.19 13.79 13.78 13.08
  • 1h at RT

Inocculation of 5 ml liquid culture (JN)

  • 4 clones of Gibson assembly of pIG15_804 in LB_cml
  • 4 clones of Gibson assembly of pET_804 in LB_amp
  • 2 clones of each ILS ligation in LB_cml

2015/07/31

Mini-prep (LS)

Inocculation of plasmids for interlab study (LS)

Test digest of pET_804 and pIG15_804 (RE)

pET_804:

  • 1 µl HindIII
  • 1 µl PstI-HF
  • 2 µl CutSmart
  • 5 µl DNA
  • 11 µl dH2O
  • expected fragment sizes: 1700 and 4100 bp

pIG15_804:

  • 1 µl EcoRI
  • 1 µl PstI-HF
  • 2 µl CutSmart
  • 5 µl DNA
  • 11 µl dH2O
  • expected fragment sizes: 800 and 2300 bp

analysis on 1% agarose gel:

Test digest of pET_804 and pIG15_804; all constructs show the expected fragment sizes :)

–> clone 3 and 4 of each construct are send in for sequencing